BACKGROUND: Syphilis spirochete Treponema pallidum ssp. pallidum remains the enigmatic pathogen, since no virulence factors have been identified and the pathogenesis of the disease is poorly understood. Increasing rates of new syphilis cases per year have been observed recently.

RESULTS: The genome of the SS14 strain was sequenced to high accuracy by an oligonucleotide array strategy requiring hybridization to only three arrays (Comparative Genome Sequencing, CGS). Gaps in the resulting sequence were filled with targeted dideoxy-terminators (DDT) sequencing and the sequence was confirmed by whole genome fingerprinting (WGF). When compared to the Nichols strain, 327 single nucleotide substitutions (224 transitions, 103 transversions), 14 deletions, and 18 insertions were found. On the proteome level, the highest frequency of amino acid-altering substitution polymorphisms was in novel genes, while the lowest was in housekeeping genes, as expected by their evolutionary conservation. Evidence was also found for hypervariable regions and multiple regions showing intrastrain heterogeneity in the T. pallidum chromosome.

CONCLUSION: The observed genetic changes do not have influence on the ability of Treponema pallidum to cause syphilitic infection, since both SS14 and Nichols are virulent in rabbit. However, this is the first assessment of the degree of variation between the two syphilis pathogens and paves the way for phylogenetic studies of this fascinating organism.

CLONEPICKER is a software pipeline that integrates sequence data with BAC clone fingerprints to dynamically select a minimal overlapping clone set covering the whole genome. In the Rat Genome Sequencing Project (RGSP), a hybrid strategy of "clone by clone" and "whole genome shotgun" approaches was used to maximize the merits of both approaches. Like the "clone by clone" method, one key challenge for this strategy was to select a low-redundancy clone set that covered the whole genome while the sequencing is in progress. The CLONEPICKER pipeline met this challenge using restriction enzyme fingerprint data, BAC end sequence data, and sequences generated from individual BAC clones as well as WGS reads. In the RGSP, an average of 7.5 clones was identified from each side of a seed clone, and the minimal overlapping clones were reliably selected. Combined with the assembled BAC fingerprint map, a set of BAC clones that covered >97% of the genome was identified and used in the RGSP.

A fosmid library representing 10-fold coverage of the Histoplasma capsulatum G217B genome was used to construct a restriction-based physical map. The data obtained from three restriction endonuclease fingerprints, generated from each clone using BamHI, HindIII, and PstI endonucleases, were combined and used in FPC for automatic and manual contig assembly builds. Concomitantly, a whole-genome shotgun (WGS) sequencing of paired-end reads from plasmids and fosmids were assembled with PCAP, providing a predicted genome size of up to 43.5 Mbp and 17% repetitive DNA. Fosmid paired-end sequences in the WGS assembly provide anchoring information to the physical map and result in joining of existing physical map contigs into 84 clusters containing 9551 fosmid clones. Here, we detail mapping the Histoplasma capsulatum genome comprehensively in fosmids, resulting in an efficient paradigm for de novo sequencing that uses a map-assisted whole genome shotgun approach.

As part of the effort to sequence the genome of Rattus norvegicus, we constructed a physical map comprised of fingerprinted bacterial artificial chromosome (BAC) clones from the CHORI-230 BAC library. These BAC clones provide approximately 13-fold redundant coverage of the genome and have been assembled into 376 fingerprint contigs. A yeast artificial chromosome (YAC) map was also constructed and aligned with the BAC map via fingerprinted BAC and P1 artificial chromosome clones (PACs) sharing interspersed repetitive sequence markers with the YAC-based physical map. We have annotated 95% of the fingerprint map clones in contigs with coordinates on the version 3.1 rat genome sequence assembly, using BAC-end sequences and in silico mapping methods. These coordinates have allowed anchoring 358 of the 376 fingerprint map contigs onto the sequence assembly. Of these, 324 contigs are anchored to rat genome sequences localized to chromosomes, and 34 contigs are anchored to unlocalized portions of the rat sequence assembly. The remaining 18 contigs, containing 54 clones, still require placement. The fingerprint map is a high-resolution integrative data resource that provides genome-ordered associations among BAC, YAC, and PAC clones and the assembled sequence of the rat genome.

Strategies for assembling large, complex genomes have evolved to include a combination of whole-genome shotgun sequencing and hierarchal map-assisted sequencing. Whole-genome maps of all types can aid genome assemblies, generally starting with low-resolution cytogenetic maps and ending with the highest resolution of sequence. Fingerprint clone maps are based upon complete restriction enzyme digests of clones representative of the target genome, and ultimately comprise a near-contiguous path of clones across the genome. Such clone-based maps are used to validate sequence assembly order, supply long-range linking information for assembled sequences, anchor sequences to the genetic map and provide templates for closing gaps. Fingerprint maps are also a critical resource for subsequent functional genomic studies, because they provide a redundant and ordered sampling of the genome with clones. In an accompanying paper we describe the draft genome sequence of the chicken, Gallus gallus, the first species sequenced that is both a model organism and a global food source. Here we present a clone-based physical map of the chicken genome at 20-fold coverage, containing 260 contigs of overlapping clones. This map represents approximately 91% of the chicken genome and enables identification of chicken clones aligned to positions in other sequenced genomes.

Fingerprinted clone physical maps have proven useful in various applications, supporting both whole-genome and region-specific DNA sequencing as well as gene cloning studies. Fingerprint maps have been generated for several genomes, including those of human, mouse, rat, the nematodes Caenorhabditis elegans and Caenorhabditis briggsae, Arabidopsis thaliana and rice. Fingerprint maps of other genomes, including those of fungi, bacteria, poplar, and the cow, are being generated. The increasing use of fingerprint maps in genomic research has spawned a need in the research community for intuitive computer tools that facilitate viewing of the maps and the underlying fingerprint data. In this report we describe a new Java-based application called iCE (Internet Contig Explorer) that has been designed to provide views of fingerprint maps and associated data. Users can search for and display individual clones, contigs, clone fingerprints, clone insert sizes and markers. Users can also load into the software lists of particular clones of interest and view their fingerprints. iCE is being used at our Genome Centre to offer up to the research community views of the mouse, rat, bovine, C. briggsae, and several fungal genome bacterial artificial chromosome (BAC) fingerprint maps we have either completed or are currently constructing. We are also using iCE as part of the Rat Genome Sequencing Project to manage our provision of rat BAC clones for sequencing at the Human Genome Sequencing Center at the Baylor College of Medicine.

Here we describe software tools for the automated detection of DNA restriction fragments resolved on agarose fingerprinting gels. We present a mathematical model for the location and shape of the restriction fragments as a function of fragment size, with model parameters determined empirically from "marker" lanes containing molecular size standards. Automated identification of restriction fragments involves several steps, including: image preprocessing, to put the data in a form consistent with a linear model; marker lane analysis, for determination of the model parameters; and data lane analysis, a procedure for detecting restriction fragment multiplets while simultaneously determining the amplitude curve that describes restriction fragment amplitude as a function of mobility. In validation experiments conducted on fingerprinted and sequenced Bacterial Artificial Chromosome (BAC) clones, sensitivity and specificity of restriction fragment identification exceeded 96% on restriction fragments ranging in size from 600 base pairs (bp) to 30,000 bp. The integrated suite of software tools, written in MATLAB and collectively called BandLeader, is in use at the BC Cancer Agency Genome Sciences Centre (GSC) and the Washington University Genome Sequencing Center, and has been provided to the Wellcome Trust Sanger Institute and the Whitehead Institute. Employed in a production mode at the GSC, BandLeader has been used to perform automated restriction fragment identification for more than 850,000 BAC clones for mouse, rat, bovine, and poplar fingerprint mapping projects.

The human genome is by far the largest genome to be sequenced, and its size and complexity present many challenges for sequence assembly. The International Human Genome Sequencing Consortium constructed a map of the whole genome to enable the selection of clones for sequencing and for the accurate assembly of the genome sequence. Here we report the construction of the whole-genome bacterial artificial chromosome (BAC) map and its integration with previous landmark maps and information from mapping efforts focused on specific chromosomal regions. We also describe the integration of sequence data with the map.

We constructed a bacterial artificial chromosome (BAC)-based physical map of chromosomes 2 and 3 of Drosophila melanogaster, which constitute 81% of the genome. Sequence tagged site (STS) content, restriction fingerprinting, and polytene chromosome in situ hybridization approaches were integrated to produce a map spanning the euchromatin. Three of five remaining gaps are in repeat-rich regions near the centromeres. A tiling path of clones spanning this map and STS maps of chromosomes X and 4 was sequenced to low coverage; the maps and tiling path sequence were used to support and verify the whole-genome sequence assembly, and tiling path BACs were used as templates in sequence finishing.